Download Chromoplast Differentiation: Current Status and

Chromoplast Differentiation: Current Status and Perspectives
1Université de Toulouse, INP-ENSA Toulouse, Génomique et Biotechnologie des Fruits, Avenue de l'Agrobiopole BP 32607, CastanetTolosan F-31326, France
2INRA, Génomique et Biotechnologie des Fruits, Chemin de Borde Rouge, Castanet-Tolosan, F-31326, France
3Universidade de São Paulo, Faculdade de Ciências Farmacêuticas, Depto. de Alimentos e Nutrição Experimental, Av. Prof. Lineu Prestes
580, bl 14, 05508-000, São Paulo, Brazil
4These authors contributed equally to this work
*Corresponding author: Email, [email protected]; Tel, +33 534 32 38 64; Fax, +33 534 32 38 73
(Received 21 July 2010; Accepted 18 August 2010)
Chromoplasts are carotenoid-accumulating plastids conferring color to many flowers and fruits as well as to some
tubers and roots. Chromoplast differentiation proceeds
from preexisting plastids, most often chloroplasts. One of the
most prominent changes is remodeling of the internal membrane system associated with the formation of carotenoidaccumulating structures. During the differentiation process
the plastid genome is essentially stable and transcriptional
activity is restricted. The buildup of the chromoplast for
specific metabolic characteristics is essentially dependent
upon the transcriptional activity of the nucleus. Important
progress has been made in terms of mediation of the
chloroplast-to-chromoplast transition with the discovery of
the crucial role of the Or gene. In this article we review recent
developments in the structural, biochemical and molecular
aspects of chromoplast differentiation and also consider the
reverse differentiation of chromoplasts into chloroplast-like
structures during the regreening process occurring in some
fruit. Future perspectives toward a full understanding of
chromoplast differentiation include in-depth knowledge of
the changes occurring in the plastidial proteome during
chromoplastogenesis, elucidation of the role of hormones
and the search for signals that govern the dialog between
the nuclear and the chromoplastic genome.
Keywords: Carotenoids synthesis • Chloroplast-to-chromoplast
transition • Chromoplast metabolism • Chromoplast structure
• Plastid genome • Regreening • Thylakoid breakdown
Abbreviations: ABA, abscisic acid; ARC5, accumulation and
replication of chloroplasts5; ARF4, auxin response factor;
CHRC, plastoglobulin carotenoid-associated protein; DDB1,
UV-damaged DNA-binding protein 1; DET1, deetiolated 1;
GFP, green fluorescent protein; MFP1, thylakoid-associated
DNA-binding protein; ORFs, open reading frames; PVD1, plastid
division1; PVD2, plastid division 2; ROS, reactive oxygen
species.
Mini Review
Isabel Egea1,2,4, Cristina Barsan1,2,4, Wanping Bian1,2, Eduardo Purgatto3, Alain Latché1,2,
Christian Chervin1,2, Mondher Bouzayen1,2 and Jean-Claude Pech1,2,*
Introduction
During evolution, higher plants have adopted strategies to
attract insects and mammals so as to facilitate flower pollination and seed dispersal. One of these strategies has been the
development of bright colors, most often within a type of plastid called chromoplasts. Chromoplasts are responsible for the
yellow, orange and red colors of many flowers and fruits. They
are also present in some roots, such as carrots, and tubers such
as sweet potatoes. Plastids are typical organelles unique to
lower and higher plants that originate from the endosymbiotic
integration of a photosynthetic prokaryote, cyanobacterium,
into a eukaryotic ancestor of algae. The ancestors of plastids,
chloroplasts, have diversified into a variety of other plastid
types, including chromoplasts, to carry out specialized functions in nonphotosynthetic organs (Pyke 2007). Among nonphotosynthetic plastids, chromoplasts have received the most
attention, as they accumulate pigments that are essential for
the sensory quality of horticultural products. Since most of the
pigments present in chromoplasts are carotenoids, the biochemistry and molecular biology of chromoplast differentiation has been largely devoted to the biochemistry and molecular
biology of carotenoid formation (Camara et al. 1995, Bramley
2002). However, despite strong specialization, nonphotosynthetic plastids also carry out many other functions either
specific to or remnants of chloroplastic functions in flowers
(Tetlow et al. 2003) and fruits (Büker et al. 1998, Bouvier and
Camara 2007). Biochemical and structural events during
chromoplast differentiation have been reviewed, either specifically (Marano et al. 1993, Ljubesic et al. 1991) or within general
papers on nongreen plastids (Thomson and Whatley 1980) and
on plastid differentiation (Waters and Pyke 2004, Lopez-Juez
2007). Since then, novel information on the specific metabolic
capacities of chromoplasts has been generated using highthroughput transcriptomic (Kahlau and Bock 2008) and proteomic approaches (Siddique et al. 2006, Barsan et al. 2010).
Plant Cell Physiol. 51(10): 1601–1611 (2010) doi:10.1093/pcp/pcq136, available online at www.pcp.oxfordjournals.org
© The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 51(10): 1601–1611 (2010) doi:10.1093/pcp/pcq136 © The Author 2010.
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This review focuses on recent data on the structural and
molecular events occurring during the differentiation of
chromoplasts to better understand how chromoplasts acquire
their specific metabolic characteristics.
Diversity of chromoplast structures
There is great variation in the morphology of chromoplasts,
particularly in the structures that contain carotenoids. A classification into globular, membranous, tubular, reticuloglobular
and crystalline has been proposed, although there is generally
more than one type of pigment-containing body in a chromoplast (Ljubesic et al. 1991). Reticulotubular structures made
of a reticulum of tubules are abundant in saffron, bananas
and Cucurbita maxima, but vesicles and globules also coexist
(Grilli-Caiola and Canini 2004). Some chromoplasts accumulate
carotenoids as large crystals inside the lumina of thylakoid-like
structures, such as in carrot roots and daffodil petals, and
plastoglubuli containing small amounts of carotenoids are also
present, although in small size and number (Kim et al. 2010).
Mango fruit chromoplasts contain large and numerous globules as well as a network of tubular membranes. They can be
considered as both the globular and reticulotubular types
(Vasquez-Caicedo et al. 2006). Tomato fruit accumulates carotenoids, predominantly in the form of lycopene crystalloids
in membrane-shaped structures (Harris and Spurr 1969).
Chromoplasts of red pepper are characterized by a large number
of globules with fibrillar extensions of carotenoid (Laborde and
Spurr 1973). Different types of chromoplasts may coexist in the
same organ. For instance, in Thunbergia alata flowers, mesophyll cells harbor chromoplasts of the tubulous type almost
exclusively, while adaxial epidermal cells contain, in addition to
the tubules, membrane and tubular reticulum structures
(Ljubesic et al. 1996). A detailed supramolecular organization of
the carotenoid-protein bodies of red pepper was described
by Deruere et al. (1994), showing that the carotenoids, in association with tocopherols and quinines, are sequestered in the
central core and are surrounded by a layer of polar lipids, which
in turn are surrounded by an outer layer of the plastoglobulin
fibrillin. In fact, fibrillin, which is highly expressed in ripening
fruit, allows the sequestration of lycopene in the form of crystals within membrane structures. The sequestration prevents
the otherwise detrimental effects of excess of carotenoids on
cellular functions. Fibril initiation occurs in the plastoglobule.
In chloroplasts where the level of carotenoids is low, the
lipid:protein ratio is sufficient for the sequestration of carotenoids. The type of carotenoid-containing bodies therefore
depends upon the lipid:protein ratio and the presence of proteins facilitating the assembly of carotenoids, such as fibrillin.
Chromoplasts that accumulate pigments during fruit
ripening and flower development are functionally different
from senescence-derived plastids. The yellow color of senescent
plastids is due to the disappearance of chlorophyll and
retention of carotenoids in the absence of de novo carotenoid
biosynthesis. In addition, contrary to chromoplasts, they
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undergo an extensive loss of plastidic DNA and are designed as
gerontoplasts (Matile 2000).
Changes in structure, morphology
and composition of the plastid during
chromoplast formation
Changes in morphology and chlorophyll-carotenoids
balance during chromoplast differentiation
Insights into the morphology of plastid differentiation and
chlorophyll breakdown have been provided by confocal
microscopy coupled with the plastid-located green fluorescent
protein (GFP; Köhler and Hanson 2000, Waters et al. 2004,
Forth and Pyke 2006). Pericarp cells in young green tomato
fruit have a large number of regular-size plastids containing
both chlorophyll and GFP, visualized by red autofluorescence
and green fluorescence, respectively. As fruits ripen, the red
fluorescence of plastids decreases in relation to chlorophyll
degradation. Fully ripe pericarp cells possess a large number
of chromoplasts, appearing as green due to the exclusive
fluorescence of GFP in the absence of chlorophyll (Forth and
Pyke 2006). The plastid size varies from the mature green to
the fully ripe stages, chromoplasts being smaller than chloroplasts. At the breaker stage, plastids show considerable
intracellular variability in size and differentiation status. The
chloroplast–chromoplast transition events are presumably
not simultaneous throughout the fruit or even within a cell,
leading to a heterogeneous population of plastids. In addition,
there are consistent differences in plastid size and appearance
between inner mesocarp and outer mesocarp cells of tomato
fruit. Chromoplasts of the outer mesocarp have an oblong,
needle-like appearance, whereas chromoplasts in the inner
mesocarp are much larger and have an ovoid shape
(Waters et al. 2004).
The transition from chloroplast to chromoplast can be
visualized during the ripening process by exploiting the autofluorescence of chlorophyll and carotenoids of purified plastid
fractions (Fig. 1). At the mature green stage, all plastids are
chloroplasts and the emitted fluorescence gives a red color
due to the dominance of chlorophyll (Fig. 1A). At the breaker
stage, the population of plastids is highly heterogeneous, but
by using adapted isolation procedures, intermediate chlorochromoplasts can be obtained containing both chlorophyll
and carotenoids so that the emitted fluorescence gives
a yellowish color due to the merging of red (chlorophyll) and
green (carotenoid) fluorescence (Fig. 1B). At the fully ripe stage,
only fully developed chromoplasts are present, and appear
green, corresponding to the autofluorescence of carotenoids
(Fig. 1C).
Preexisting plastids from which chromoplasts
originate
Different forms of plastids can be generated by interconversions of preexisting plastid types. A cycle of plastid development
Plant Cell Physiol. 51(10): 1601–1611 (2010) doi:10.1093/pcp/pcq136 © The Author 2010.
Chromoplast differentiation
Fig. 1 Confocal images of chloroplast (A), chloroplast initiating transition (B) and mature chromoplast (C) suspensions isolated from mature
green (D), breaker (E) and fully ripe (F) tomatoes. Images are overlays of chlorophyll autofluorescence and carotenoid autofluorescence emitted
at wavelengths between 740 and 750 nm (red) and between 500 and 510 nm (green), respectively, when they are excited using the 488 nm line
from the argon laser. Structures containing mainly chlorophyll appear red, those containing only carotenoid appear green, and those containing
both chlorophyll and carotenoid appear orangey red/yellow. Scale bars = 16 µm.
interrelationships has been suggested (Whatley 1978). Chloroplast differentiation from proplastids, under the control of
light, is one of the best-known interconversions (Lopez-Juez
and Pyke 2005). Chromoplasts may arise directly from proplastids (e.g. in carrot roots; Ben-Shaul and Klein 1965), indirectly
from chloroplasts (e.g. in ripening fruit; Bathgate et al. 1985)
or from amyloplasts (e.g. in saffron flowers; Grilli-Caiola and
Canini 2004; or tobacco floral nectaries; Horner et al. 2007). An
interesting example of plasticity exists in Arum italicum berry
fruit, where the various steps of maturation and ripening are
associated with a sequence of transitions involving amyloplast,
chloroplast and chromoplast (Bonora et al. 2000).
Analysis of plastid division in tomato fruit revealed that
the majority of plastid division by binary fission occurs during
the fruit enlargement stages when the plastids are present as
chloroplasts. The plastid number remains fairly constant once
ripening begins (Cookson et al. 2003). Replication of chromoplasts is occasionally observed, such as in pepper fruit (Leech
and Pyke 1988) and Forsythia suspensa petals (Sitte 1987).
Replication by budding and fragmentation has also been
observed in the suffulta mutants, in which a heterogeneous
population of plastids exist (Forth and Pyke 2006). In agreement with the absence or low rate of division in regular tomato
chromoplasts, only a few members of the plastid division
machinery have been encountered in the proteome (Barsan et al.
2010). Several homologs of the 3 FtsZ proteins of Arabidopsis
have been detected, but the other parts of the plastid
division machinery (Pyke 2007), such as ACCUMULATION
AND REPLICATION OF CHLOROPLASTS5 (ARC5), PLASTID
DIVISION1 (PVD1) and PLASTID DIVISION2 (PVD2), were
absent.
Internal membrane remodeling during
chromoplast formation
Electron microscopy studies carried out in red pepper by Spurr
and Harris (1968) have shown that remodeling of the internal
membrane system starts with lysis of the grana and the intergranal thylakoids. Some small and loosely aggregated groups
of initial thylakoids still persist at advanced stages of ripening.
In parallel, new membrane systems are formed consisting in
organized membrane complexes called thylakoid plexus by
Spurr and Harris (1968) and thylakoid sheets. These early observations are consistent with most recent data (Simkin et al.
2007) showing that during the chloroplast–chromoplast conversion in tomato fruit, the thylakoid disassembly is associated
with the synthesis of new membranes that are the site for the
formation of carotenoid crystals. These newly synthesized
membranes do not derive from the thylakoids, but rather from
Plant Cell Physiol. 51(10): 1601–1611 (2010) doi:10.1093/pcp/pcq136 © The Author 2010.
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vesicles generated from the inner membrane of the plastid
(Simkin et al. 2007).
The loss of thylakoid integrity revealed by ultrastructure
studies corresponds to a late event of chromoplast development that is visible well after the loss of thylakoid-associated
metabolic functions. For instance, in the flower bud of
Lilium longiflorum, the rapid decline of photosynthetic activity
during the chloroplast–chromoplast transition occurs well
before any observable loss of thylakoid integrity and reduction
of chlorophyll (Clément et al. 1997). The metabolic machineries
are not affected at the same rate. Within the photosynthetic
apparatus, photosystem II integrity was preserved longer than
the rest of the machinery (Juneau et al. 2002). Interestingly,
the loss of thylakoid integrity during tomato fruit ripening is
associated with a large decrease of a thylakoid-associated
DNA-binding protein, MFP1, which is supposed to participate
in the development of the thylakoid membrane (Jeong et al.
2003).
Role of plastoglobules and plastoglobulins
in the storage of carotenoids
During the chloroplast–chromoplast transition, an increase in
the size and number of plastoglobuli is generally observed
(Harris and Spurr 1969). Microscopy studies demonstrated
that plastoglobules arise from a blistering of the stroma-side
leaflet of the thylakoid membrane, predominantly along highly
curved margins (Austin et al. 2006).
There is experimental evidence that plastoglobulins participate in the sequestration of carotenoids and in the biogenesis
of chromoplasts (reviewed by Bréhélin and Kessler 2008). The
observation that suppression in tomato plants of the plastoglobulin carotenoid-associated protein (CHRC) results in 30%
reduction of carotenoids in tomato flowers provided the first
evidence for the role plastoglobulin chromoplast differentiation (Leitner-Dagan et al. 2006). Other evidence included the
overexpression in tomato of a pepper plastoglobulin, fibrillin,
that caused an increase in carotenoid and carotenoid-derived
flavor volatiles (Simkin et al. 2007). In addition, the loss of
thylakoids was delayed during the chloroplast–chromoplast
transition and the plastids showed a typical chromoplastic
zone contiguous with a preserved chloroplastic zone. It is concluded that fibrillin plays a role in thylakoid disorganization
during chromoplast formation.
Plastoglobules not only act as lipid storage bodies, but they
also participate in some metabolic pathways (Bréhélin and
Kessler 2008). Analysis of the proteome of red pepper plastoglobules indicated the presence of several proteins involved in
the synthesis of carotenoids, including ζ-carotene desaturase,
lycopene β-cyclase and two β-carotene β-hydroxylases
(Ytterberg et al. 2006). Also, ζ-carotene desaturase has been
detected in the proteome of tomato chromoplasts (Barsan
et al. 2010).
The chloroplast–chromoplast transition is shown in Fig. 2,
in which remodeling of the internal membrane system and
formation of carotenoid storage structures are represented.
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Changes in stromule morphology during
chromoplastogenesis
Stromules are motile protrusions emanating from the plastid
membrane into the cytoplasm. Microscopy techniques coupled
with GFP have revealed that the importance of stromules
generally increases with the progress of fruit ripening in tomato
(Waters et al. 2004). However, there are some differences
between tomato tissues. Long stromules are associated with
plastids that are further apart, whereas short stromules are
present in cells with a high density of plastids. In the outer
mesocarp, where cells have a high density of plastids, stromules
are short and form a complex chromoplast network (Pyke and
Howells 2002). In the inner mesocarp, the density of plastids is
lower and stromules are longer and their number and length
increases during ripening (Waters et al. 2004). Once the fruit
begins to ripen, stromules increase in number and length, at
least in the inner mesocarp, probably to provide a greater
import area for novel proteins (Kwok and Hanson 2004),
particularly those involved in carotenoid biosynthesis and
chromoplast differentiation. Sometimes, free broken stromules
detached from the plastid appear throughout the cytoplasm of
green fruit as small vesicles containing only GFP. They may have
the potential to develop into full chromoplasts (Waters et al.
2004). The green flesh mutation, in which plastid differentiation
is incomplete, and the rin mutation, in which the ripening process is blocked, result in a reduction of stromule formation
(Waters et al. 2004).
Characteristics and stability of the
plastid genome during differentiation
into chromoplasts
The plastid genome (plastome) of the tomato fruit has the
same basic characteristics as the majority of the plastomes that
have been sequenced. The size of the tomato fruit plastome is
155,461 bp and comprises a large and a small single-copy region
intercalated by two inverted repeats, IRa and IRb (Kahlau et al.
2006). Annotation indicated the presence of 114 genes and
conserved open reading frames (ORFs) divided into three
major categories (Sugiura 1992): (i) photosystem-related genes;
(ii) genetic system genes, including genes encoding ribosomal
proteins, tRNA and a plastid RNA polymerase; and (iii) the
hypothetical chloroplast reading frames (ycfs), a group of
conserved sequences, some of them of unknown function, but
essential for plastids activity (Ravi et al. 2008).
Comparison by restriction enzyme analysis of the DNA of
chloroplasts from leaves and chromoplasts from tomato fruit
revealed the absence of rearrangements, losses or gains (Hunt
et al. 1986). Subtle changes in DNA, such as increased methylation of cytosine, have been suggested upon analysis by liquid
chromatography of tomato chromoplasts (Kobayashi et al. 1990).
However, this observation was not confirmed by Marano and
Carrillo (1991), who found that the patterns of DNA methylation assessed after restriction and hybridization analysis with
Plant Cell Physiol. 51(10): 1601–1611 (2010) doi:10.1093/pcp/pcq136 © The Author 2010.
Chromoplast differentiation
Chloroplast
Stromule
Chromoplast
Starch
grain
1
Cristalloid carotenoid
structures
7
6
5
Ribosomes
Plastoglobules
4
Thylakoid
remnants
6
Carotenoids
Circular
DNA
4
Membranous sac
Granum
3
Stromule
Lamella
Lumen
Plastoglbules
Thylakoid
Envelope
Stroma
Chromoplast internal membrane
7
Fig. 2 Schematic representation of the chloroplast–chromoplast transition. The scheme shows the breakdown of starch granules (1) and of grana
and thylakoids (2); the synthesis of new membrane structures form the inner membrane envelope of the plastid (3) leading to the formation
carotenoid-rich membranous sacs (4); the increase in the number and size of plastoglobules (5); the appearance of carotenoid-containing
crystalloids (6); and the increase in the number of protrusions emanating from the plastid envelope, called stromules (7).
DNA probes did not differ significantly between chloroplasts of
mature green tomatoes and chromoplasts of red ripe fruit.
Since structural and methylation changes in DNA have not
been firmly established, their role in plastid switching remains
uncertain.
Importance of transcriptional and translational
activity during chromoplast differentiation
The expression pattern of few plastid localized genes has been
studied. As expected, genes involved in carotenoid biosynthesis, such as lycopene β-cyclase (CYCB), are up-regulated during
chromoplast formation in many plants, including citrus fruit
(Alquezar et al. 2009); the wild species of tomato, Solanum
habrochaites (Dalal et al. 2010); safron (Ahrazem et al. 2010);
papaya fruit (Blas et al. 2010); and carrot (Chen et al. 2001).
On the contrary, genes involved in photosynthetic activity are
generally down-regulated during chromoplast formation
(Cheung et al. 1993). Surprisingly, up-regulation of the large
subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase
and the 32 kD photosystem II quinone binding protein
genes has been observed in chromoplasts of squash fruits
(Cucurbitae pepo; Obukosia et al. 2003), indicating that the
expression pattern of these photosystem genes may be
regulated independently from the plastid differentiation
processes.
A comprehensive study of chromoplastic transcriptome was
carried out by Kahlau and Bock (2008) showing that the global
transcriptional activity remains almost the same during chromoplast differentiation, except for a limited number of genes,
including (i) accD, which encodes a subunit of the acetyl-CoA
carboxylase involved in fatty acid biosynthesis; (ii) trnA (tRNAALA); and (iii) rpoC2 (RNA polymerase subunit). During fruit
ripening a reduction of translational activity has been observed
by comparison of polysome-associated plastidial mRNA levels
between fruit chloroplasts and chromoplasts. Rather than
a decrease in transcription, plastid translation appears to be
the main factor that contributes to down-regulation of chromoplast proteins during fruit plastid differentiation. Another
line of evidence to support this hypothesis is that the activity
of both the nuclear-encoded and plastid-encoded RNA polymerases undergoes little change during the transition. Likewise,
RNA splicing activity of the plastid, a possible mechanism
contributing to the regulation of gene expression, exhibits
insignificant changes during tomato fruit ripening. In any case,
transcriptional and translational activities of the plastid make
a limited contribution to chromoplast differentiation. The large
majority of the proteins present in the plastid are encoded
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by nuclear genes so that transcriptional activity in the nucleus
and translocation of proteins into the plastid are of primary
importance for the buildup of chromoplast metabolism.
Proteins related to the biosynthesis of fatty acids, amino acids,
carotenoids, vitamins, hormones, aroma volatiles and other
substances have been encountered in the chromoplastic proteome of tomato (Barsan et al. 2010). These proteins participate in giving the fruit important sensorial characteristics
such as color and aroma. Many of the corresponding genes
are regulated by the plant hormone ethylene and therefore
participate in the transcriptional regulation of the fruit
ripening process in general (Giovannoni 2001, Pirrello et al.
2009). As shown in the following paragraph, some of the
nuclear-localized genes play a crucial role in chromoplast
differentiation.
Genes involved in chromoplast
differentiation and development of
carotenoid storage structures
Due to increased expression during the choloroplast–
chromoplast transition, some genes have been suspected to
play a role in chromoplastogenesis. Such is the case for the
early light-inducible protein (ELIP) gene, which has homology
with light-harvesting complex proteins and whose expression
is high during the breaker/turning ripening stages in tomato.
However, no direct evidence for a role of ELIP or other genes
in chromoplast differentiation had been provided until the
discovery of the cauliflower Or gene (Lu et al. 2006). The
dominant mutation Or confers an orange pigmentation
with accumulation of β-carotene mostly in the inflorescence
of cauliflower without significantly affecting the expression of
carotenoid biosynthetic genes (Li et al. 2001). Chromoplasts
differentiate in the OR mutant and develop membranous
inclusions of carotenoids resembling those of carrot roots.
In addition, there was an arrest in plastid division, and for this
reason only one or two chromoplast are present in the affected
cell (Paolillo et al. 2004). Chromoplast differentiation occurs
mostly in the inflorescence tissues, but not in the leaves,
suggesting that tissue-specific expression is regulated at the
transcriptional or posttranscriptional levels. The Or transgene
introduced in a tuber-specific manner into potato induces
a sharp increase in the accumulation of carotenoids, again
without affecting the expression of endogenous carotenoid
biosynthetic genes (Lu et al. 2006, Lopez et al. 2008). The Or
gene is nuclear-localized and encodes a DnaJ-like cochaperone
containing a cysteine-rich domain lacking the J-domain
(Lu et al. 2006). The role of the DnaJ proteins is to interact
with Hsp70 chaperones to perform protein folding, assembly,
disassembly and translocation. The absence of phenotype
upon RNAi silencing suggests that Or is not a loss-of-function
mutation, and putative interaction with Hsp70 chaperones
indicates that it might be a dominant-negative mutation
(Giuliano and Diretto 2007). Altogether, these data show
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that the Or gene is not directly involved in carotenoid biosynthesis, but rather causes a metabolic sink for carotenoid
accumulation by inducing the formation of chromoplasts
(Li and van Eck 2007).
The role of some genes in the formation of carotenoid storage structures has been explored. Overexpression of phytoene
synthase gene causes carotenoid crystal formation on nongreen
tissues of Arabidopsis, but not in green tissues, indicating
a fundamental difference in carotenoid storage mechanisms
(Maass et al. 2009). Therefore the sequestration of carotenoids
into crystals resulting from high activity of phytoene synthase
can happen in the absence of a chromoplast developmental
program, such as in Arabidopsis, as a consequence of enhanced
carbon flux through the pathway. High phytoene synthase has
also been associated with β-carotene accumulation in orange
carrot roots (Maass et al. 2009).
Metabolic activities of chromoplasts
The metabolic activity of chromoplasts has already been
reviewed (Neuhaus and Emes 2000, Bouvier and Camara 2007).
Here we provide an overview of the main features and include
some recent data. When chromoplasts derive from chloroplasts, the most obvious biochemical change is the loss of
chlorophyll and photosynthetic activity associated with the
down-regulation of photosynthetic gene expression (Piechulla
et al. 1985). Another major feature of chromoplast metabolism
is the accumulation of pigments. Several reviews have been
dedicated to the biosynthesis of carotenoids in fruits and flowers (Bramley 2002, Fraser and Bramley 2004, Lu and Li 2008).
However, they are also the site for the synthesis of sugars,
starches, lipids, aromatic compounds, vitamins (riboflavin,
folate, tocopherols) and hormones (Neuhaus and Emes 2000,
Barsan et al. 2010). For sustaining biosynthetic activities, sugars
are imported from the cytosol by a plastid-localized glucose
transporter (Bouvier and Camara 2007), but the use of endogenous sugars resulting from starch degradation cannot be
excluded. Proteins of starch biosynthesis and degradation
remain present in tomato chromoplasts (Barsan et al. 2010).
Calvin cycle enzymes have been measured in plastids isolated
from sweet pepper, and their activities were generally greater in
chromoplasts than in chloroplasts (Thom et al. 1998). In tomato,
the activity of Calvin cycle enzymes has also been observed
(Obiadalla-Ali et al. 2004). In association with the persistence
of the active oxidative phosphate pathway (Tetlow et al. 2003,
Bouvier and Camara 2007, Barsan et al. 2010), adenosine
triphosphate (ATP) and reducing power are produced that
also participate in sustaining the metabolic activities of chromoplasts. Another interesting feature of chromoplasts is the
presence of highly active antioxidant system. The level of glutathione and ascorbate in the plastids isolated from pepper fruit
increase during fruit ripening in parallel with the activity of the
enzymes of the ascorbate glutathione cycle and superoxide
dismutase (Marti et al. 2009). High activity of the antioxidant
system in the chromoplasts could play a role in protecting
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Chromoplast differentiation
plastid components such as carotenoids against oxidation, but
also in mediating signaling between chromoplasts and the
nucleus. Reactive oxygen species (ROS) are thought to participate in plastid to nucleus communication (Kleine et al. 2009,
Galvez-Valdivieso and Mullineaux 2010). Plastid-generated ROS
are known to up-regulate the transcription of genes of carotenoid biosynthesis (Bouvier et al. 1998).
Reversible differentiation of chromoplasts
Reversible differentiation of plastids is another aspect of plasticity of the organelle. Preberg et al. (2008) quote a number of
situations where regreening of tissues occurs as a consequence
of redifferentiation of gerontoplasts, etioplasts or chromoplasts
into chloroplasts. The phenomenon is truly a redifferentiation
process without any evidence of de novo generation of plastids
or plastid division. In the case of chromoplasts, the best known
example of reversal to chloroplasts is that of citrus fruits
(Thomson et al. 1967), but the phenomenon also exists in other
species such in cucumber fruit (Preberg et al. 2008). Ultrastructural aspects of the reversion of chromoplasts to chloroplasts
have been described in the subepidermal layer of fruit of
Cucurbita pepo (Devide and Ljubesic 1974). During regreening,
the globular-type chromoplasts with numerous plastoglobules
and small vesicle-like fragments of thylakoids undergo a disappearance of plastoglobules and the formation of new thylakoids. Thylakoids arise from both preexisting vesicles and
from the invagination of the inner membrane of the plastid to
form grana structures, leading to normal chloroplast structure
and photosynthetic activity. Similar reconstitution of the
thylakoid system has been described recently along with more
details during redifferentiation of chloroplasts in cucumber
fruit (Preberg et al. 2008). In this case, the plastoglobules persisted during the entire process and remnants of the degraded
thylakoid system formed large membrane-bound bodies that
later participated in the reformation of thylakoids.
Light is probably the most important factor of regreening via
phytochromes, however, nutritional factors are also involved.
Warm temperatures, nitrogen fertilization and gibberellins
stimulate regreening of citrus peel, while an abundance of
sucrose tends to inhibit this process (Huff 1983). Very little
information is available on the molecular mechanisms of
reversal from chromoplasts to chloroplasts. However, there is
evidence that gibberellic acid, which stimulates the greening
process, reduces the expression of carotenoid biosynthetic
genes, phytoene synthase, phytoene desaturase and β-carotene
hydroxylase in orange flavedo (Rodrigo and Zacarias 2007).
In clementines, gibberellins and nitrate that favor regreening
reduced the expression of not only phytoene synthase, but also
of the chlorophyll-degrading gene pheophorbide a oxygenase
(Alos et al. 2006), indicating that regreening involves both
repression of carotenoid biosynthesis and reduction of chlorophyll breakdown. No information is available yet on the expression of genes involved in the biosynthesis of photosystems and
chlorophyll.
Mutants with altered chromoplast
development
We have already mentioned the Or mutant of cauliflower,
which has allowed the isolation of a gene controlling the differentiation of chromoplasts. Here we briefly examine other
mutants, mostly of tomato, showing, among other phenotypes,
altered plastid development. Despite the pleiotropic effects of
the mutation, these mutants represent useful tools for the
identification of the molecular players involved in chromoplast
biogenesis.
Compared to wild type, the natural mutants HIGH PIGMENT 1
and 2 (hp1 and hp2) have dark-green immature fruits and
accumulate higher levels of carotenoids in ripe fruits (Yen et al.
1997, Mustilli et al. 1999). The hp1 mutant codes a homolog
of the Arabidopsis UV-DAMAGED DNA-BINDING PROTEIN 1
(DDB1) protein, which is predicted to interact with the nuclear
factor DEETIOLATED 1 (DET1) (Liu et al. 2004), while the hp2
codes a tomato ortholog of Det1 (Mustilli et al 1999). Ripe fruits
of both mutants contain more and bigger chromoplasts per cell
than wild-type fruits. The product of the Det1 gene is part of
the CUL4-based E3 ubiquitin ligase complex of the proteasome
(Bernhardt et al. 2006, Wang et al. 2008). In the HIGH PIGMENT
3 mutant (hp3), which has the same phenotype as hp1 and hp2,
the level of abscisic acid (ABA) is lower than in wild-type plants,
suggesting that ABA deficiency could be an important factor
for the development of the phenotype. This hypothesis is further sustained by the analysis of two other ABA-deficient
tomato mutants, flacca and sitiens, that harbor similar alterations in plastid development (Galpaz et al. 2008).
The mutation in the locus suffulta provokes changes in the
division of plastids in tomato plants, generating cells with giant
chloroplasts but with low chlorophyll content. An unusual
process of plastid division occurs during the chloroplast–
chromoplast transition, characterized by budding and plastid
fragmentation into small vesicles. This results in a heterogeneous population of chromoplasts at different development
stages, with some of them keeping the chloroplast structure
(Forth and Pyke 2006). The molecular identity of the gene
responsible for the suffulta phenotype is unknown. It could be
a component of the plastidial division machinery or could regulate the division process. It could also participate in the differentiation of chromoplasts.
In addition to light, phytohormones have been reported to
play an important role in controlling chloroplast/chromoplast
formation and stability during tomato fruit development. It is
well known that tomato mutants and transgenic lines, impaired
in elements of the ethylene signaling transduction cascade
such as the ethylene receptor NR (Wilkinson et al. 1995), present altered pigmentation. Down-regulation of ARF4, an auxin
response factor formerly named DR12, resulted in a dark-green
phenotype and blotchy ripening of tomato fruit (Jones et al.
2002). In the ARF4 down-regulated lines, the outer pericarp
tissue displayed a higher number of chloroplasts per cell and
a dramatic increase in grana formation. Interestingly, in contrast
Plant Cell Physiol. 51(10): 1601–1611 (2010) doi:10.1093/pcp/pcq136 © The Author 2010.
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I. Egea et al.
to hp mutants, the dark-green phenotype in ARF4-inhibited
lines is confined to the fruit. The treatment of tomato fruits
with fluridone, an inhibitor of ABA synthesis, has inhibitory
effects on carotenoid accumulation (Zhang et al. 2009).
It was also reported that exogenous treatment with cytokinin
can mimic the hp mutant phenotype (Mustilli et al. 1999)
and that cytokinin hypersensitive Arabidopsis mutants show
increased chloroplast development (Kubo and Kakimoto
2000). The blotchy ripening phenotype was also induced by
ectopic expression in tomato lines of the ipt gene from the
Ti plasmid of Agrobacterium tumefaciens (Martineau et al.
1994). In this latter case, fruit displayed higher levels of
cytokinin, and during ripening the fruit exhibited an altered
phenotype, with green patches remaining within a deep red
background.
The rin tomato mutant harbors a nonfunctional MADSbox transcription factor that is essential for fruit ripening
(Vrebalov et al. 2002). The number of chromoplasts per cell in
rin fruits at the breaker stage is much higher than the wild type,
and the plastids are very small with few stromules. Whether
the RIN gene is a direct regulator of the plastid transition in
tomato fruit or the lack of ethylene synthesis in rin fruits is
responsible for the abnormal chromoplast biogenesis remains
unclear.
Green flesh and chlorophyll retainer are mutants of tomato
and pepper, respectively, that have no ability to degrade
chlorophyll during fruit ripening, but are able to synthesize
carotenoids, resulting in brown color fruits. The plastids in the
ripe fruit of these two mutants have remnants of thylacoidal
membranes and formation of plastoglobuli, suggesting that
the conversion of chloroplasts to chromoplasts is not completely concluded. The level of the carotenoids in the mutants
is lower than in wild-type fruits, and several photosystem genes,
like rbcL and cab, are up-regulated. Barry et al. (2008) indicated
the possibility that this mutation is due to an impaired gene
product linked to the chlorophyll degradation pathway.
All mutations described above show evidence that chromoplast formation is a complex event that involves not only
factors expressed during ripening, but also developmental
factors and hormones like auxin, cytokinin, ABA and ethylene.
Two processes seem to be important for normal chromoplast
biogenesis, chloroplast division and the biosynthesis of carotenoids. However, the way in which these processes are coordinated by nuclear and plastid gene expression remains unclear
and represents a challenge for future studies.
differentiation associated with the use of comparative proteomic methods represent an interesting perspective toward
the uncovering of target proteins that play a role in the chromoplast differentiation process.
Moreover, the combination of transcriptomic and proteomic data will allow identification of molecular events
that are regulated at the transcriptional, posttranscriptional
or posttranslational levels. In particular, the identification of
phosphorylation and other types of protein modifications
by proteomic analysis will provide useful information on
the levels of regulation of plastid metabolic activity during
chloroplast–chromoplast transition steps.
While a number of experimental data support the role
of phytohormones in regulating plastid differentiation and
evolution during ripening, the underlying mechanisms remain
unclear. Also, although many hormones such as ethylene,
auxin, cytokinin and ABA seem to take part in the regulation
of the chloroplast–chromoplast transition, the extent of
the cross talk between hormones to tune the process is
unknown.
It is admitted that the expression of many genes targeted to
plastids is regulated through a dialog between the nucleus and
the plastid. These signals, whether environmental (temperature, light, etc.) or developmental, are supposed to comprise
ROS, carotenoids, carbohydrates and hormones (ABA, jasmonates). The presence of a plastid–nucleus dialog is verified
by the fact that exposure of the chloroplast to tagetitoxin, a
specific inhibitor of plastidial RNA polymerase (Rapp and
Mullet 1991), or lincomycin, a specific inhibitor of plastid peptidyl transferase (Mulo et al. 2003), decreases the accumulation
of plastid-targeted nuclear transcripts. However, the contribution of these signals to the expression of specific genes is far
from being fully understood (Kleine et al. 2009). In addition,
most of the studies dealing with nucleus-to-plastid signaling
have been carried out with chloroplasts. Whether some of these
mechanisms are active in nonphotosynthetic plastids, such as
chromoplasts, remains an open question. We are still far from
a clear understanding of the dialog between the nuclear and
the plastidial genome in mediating the differentiation of chromoplasts, and thus we are far from controlling developmental
processes such as fruit ripening and flower development.
Funding
This work was supported by the Midi-Pyrénées Regional
Council [grant numbers CR060033789, CR07003760].
Future perspectives
The functional genomics tools will allow new insights into the
mechanisms of chromoplast development. As revealed by
a comprehensive survey with the new mass spectrometry
technologies, the number of proteins assigned to the chromoplast proteome (Barsan et al. 2010) is comparable to that of
the chloroplast proteome (Ferro et al. 2010). The development
of specific protocols for isolating plastids at different stages of
1608
Acknowledgements
Isabel Egea received a postdoctoral fellowship from “Fundación
Séneca” (Murcia, Spain). Cristina Barsan received a bursary
from the French Embassy in Bucharest (Romania) for a joint
“co-supervision” PhD, and Wanping Bian received a bursary
from the University of Chongqing (China) for a PhD. The participation of Eduardo Purgatto was made possible by a sabbatical
Plant Cell Physiol. 51(10): 1601–1611 (2010) doi:10.1093/pcp/pcq136 © The Author 2010.
Chromoplast differentiation
postdoctoral fellowship from the government of Brazil
(CNPq). We are indebted to Dr Alain Jauneau (IFR40, CNRS,
University of Toulouse) for his contribution to the confocal
microscopy work.
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